LOCUS       sequence-416571-        7436 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express eGFP-CCM2 with the I432D mutation in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7436)
  AUTHORS   Ball NJ, Ghimire S, Follain G, Pajari AO, Wurzinger D, Vaitkeviciute
            M, Cowell AR, Berki B, Ivaska J, Paatero I, Goult BT, Jacquemet G
  TITLE     TLNRD1 is a CCM complex component and regulates endothelial barrier 
            integrity.
  JOURNAL   J Cell Biol. 2024 Sep 2;223(9):e202310030. doi: 
            10.1083/jcb.202310030. Epub 2024 Jul 16.
  PUBMED    39013281
REFERENCE   2  (bases 1 to 7436)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Cell 
            Biol."; date: "2024-09-2"; volume: "223(9):e202310030. doi"; pages: 
            " 10.1083/jcb.202310030. Epub 2024 Jul 16"
COMMENT     Sequence Label: CCM2_I432D_eGFP
FEATURES             Location/Qualifiers
     source          1..7436
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     11..30
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(90..106)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(90..106)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(103..125)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    114..130
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(138..144,145..162,163..168))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    183..204
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(321..338)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(492..1080)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(572..591)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1251..2042,2043..2111))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     1874..1893
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2112..2216)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2291..2310)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2482..2861
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2862..3065
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3016..3036
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     3110..3129
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3110..3128
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3170..3883
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             join(3170..3172,3173..3883)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(3212..3233)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3473..3492)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3820..3841
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(5274..5291)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5280..5504
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      5550..5978
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5637..5656)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5847..5868
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5987..6007)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5992..6321
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6172..6307
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6234..6253
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             6388..7182
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(6442..6461)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     7052..7071
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    join(7356..7436,1..41)
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7393..7412)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
ORIGIN
        1 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg
       61 acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
      121 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
      181 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
      241 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
      301 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
      361 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
      421 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
      481 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
      541 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
      601 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
      661 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
      721 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
      781 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
      841 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
      901 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
      961 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     1021 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     1081 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     1141 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     1201 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     1261 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     1321 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     1381 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     1441 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     1501 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     1561 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     1621 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     1681 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     1741 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     1801 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     1861 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     1921 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     1981 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     2041 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     2101 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     2161 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     2221 tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt
     2281 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt
     2341 gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct
     2401 tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg
     2461 tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta
     2521 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
     2581 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
     2641 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
     2701 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
     2761 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
     2821 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
     2881 acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
     2941 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca
     3001 actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
     3061 gagctctctg gctaactaga gaacccactg cttactggct tatcgaaatt aatacgactc
     3121 actataggga gacccaagct ggctagcgtt taaacttaag cttgccacca tgagcaaggg
     3181 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg
     3241 ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct
     3301 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct
     3361 gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt
     3421 caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg
     3481 caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga
     3541 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
     3601 ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa
     3661 cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca
     3721 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca
     3781 gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
     3841 gaccgccgcc gggatcactc acggcatgga cgagctgtac aagggtaccg agctcggatc
     3901 catggaagaa gagggcaaga agggcaagaa acctggcatc gttagcccct tcaagcgggt
     3961 gttcctgaag ggcgaaaaga gcagagataa gaaggcccat gagaaagtga cagagagaag
     4021 acctctgcac accgtggtcc tgtccctgcc tgagcgcgtg gaacctgacc ggctgctgtc
     4081 tgattacatc gagaaagagg tgaagtacct gggccagctg acctctatcc ccggctacct
     4141 gaacccatct agccggaccg agatcctgca cttcatcgac aacgccaaga gggcccacca
     4201 actcccaggc cacctgacac aggagcacga tgccgtgctg tccctgagcg cctacaacgt
     4261 caagctggcc tggcgggatg gagaagatat tatcctcaga gtgcctatcc acgacatcgc
     4321 cgctgttagc tacgtgcggg acgacgccgc ccacctggtg gtgctgaaga ccgcccagga
     4381 ccccggcatc tctccttcac agagcctgtg cgctgagtcc agcagaggcc tgtccgcagg
     4441 ctctctaagc gagagcgccg tgggacctgt ggaagcatgc tgcctggtca tcctggccgc
     4501 cgagagcaag gtggccgctg aggaactgtg ttgcctgctg ggccaagtgt ttcaggtggt
     4561 gtacaccgaa agcaccattg acttcctgga cagagctatc ttcgacggcg cttctacacc
     4621 tacccaccac ctcagtctgc attctgacga cagctctacc aaggtggaca tcaaggaaac
     4681 ctacgaggtg gaagcctcta cattttgctt ccccgagagc gtggatgtgg gcggagctag
     4741 cccccactcc aaaaccatca gcgagtccga actgtctgcc agcgctactg agctgctgca
     4801 ggactatatg ctgacactga gaaccaagct gagcagccag gagatccagc agttcgccgc
     4861 cctgctgcac gagtacagaa acggcgccag catccacgag ttctgcatca atctgagaca
     4921 gctgtacggc gatagcagaa agttcctgct gctgggactg aggcctttca tcccagaaaa
     4981 agactcccaa cactttgaga acttcctgga aaccatcgga gtgaaggacg gccgaggcat
     5041 catcaccgac agcttcggca gacacagacg ggccctgagc accacaagca gcagcacaac
     5101 caacggcaat agagccacag gcagcagcga cgaccggagc gcccctagcg aaggcgacga
     5161 gtgggataga atgatctccg acatcagcag cgatgatgag gccctgggat gtagcatgga
     5221 ccaggacagc gcctaactcg agtctagagg gcccgtttaa acccgctgat cagcctcgac
     5281 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     5341 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     5401 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     5461 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag
     5521 aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc
     5581 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     5641 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     5701 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     5761 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     5821 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     5881 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     5941 aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag
     6001 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     6061 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     6121 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     6181 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     6241 gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt
     6301 ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat
     6361 caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct
     6421 ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc
     6481 tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc
     6541 gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc
     6601 acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg
     6661 ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag
     6721 aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc
     6781 ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt
     6841 cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc
     6901 gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc
     6961 tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg
     7021 ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag
     7081 cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg
     7141 cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg
     7201 aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct
     7261 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
     7321 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg
     7381 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactg
//
